Dengue virus, a member of the flavivirus family, imposes one of the largest social and economic burdens of any mosquito-borne viral pathogen. There is no specific treatment for infection, and control of dengue virus by vaccination has proved elusive. Several other flaviviruses are important human pathogens, including yellow fever, West Nile, tick-borne encephalitis (TBE) and Japanese encephalitis viruses (JE).
Three structural proteins (“C”, “M”, and “E”) and a lipid bilayer package the positive-strand RNA genome of flaviviruses. The core nucleocapsid protein, C, assembles with RNA on the cytosolic face of the endoplasmic reticulum membrane. The assembling core buds through the ER membrane, thereby acquiring an envelope that contains the major envelope glycoprotein, E, and the so-called precursor membrane protein, PrM. The particle passes through the secretory pathway, where a furin-like protease cleaves PrM to M in a late trans-Golgi compartment. The cleavage, which removes most of the ectodomain of PrM, releases a constraint on E and primes the particle for low-pH triggered membrane fusion. Uncleaved, immature particles are not fusion competent.
Enveloped viruses enter cells by membrane fusion. E, which mediates both receptor binding and fusion, is a so-called “class II” viral fusion protein. Two classes of viral “fusion machines” have been identified so far. Class I viral fusion proteins include those of the myxo- and paramyxoviruses (e.g., influenza), the retroviruses (e.g., HIV), and the filoviruses (e.g., Ebola). Class II fusion proteins are found in not only the flaviviruses (yellow fever, West Nile, etc.), but also the alphaviruses (Semliki Forest virus, Sindbis virus, etc. . . . ), as well as Hepatitis C. The structural characteristics of the two classes are quite different, but both accomplish the same “reaction”—viz., fusion of two lipid bilayers.
The more familiar class I fusion proteins, exemplified by the haemagglutinin (HA) of influenza virus and gp120/gp41 of HIV, have a “fusion peptide” at or near the N-terminus of an internal cleavage point. This hydrophobic and glycine-rich segment, buried in the cleaved-primed trimer of the class I fusion protein, emerges when a large-scale conformational rearrangement is triggered by low pH (in the case of HA), receptor binding (in the case of gp120/gp41), or other cell-entry related signal. The likely sequence of events that follow include an interaction of the fusion peptide with the target-cell membrane and a refolding of the trimer. The latter step brings together the fusion peptide and viral-membrane anchor, thereby drawing together the cellular and viral membranes and initiating the bilayer fusion process.
The class II proteins, found so far in flaviviruses and alphaviruses, have evolved a structurally different but mechanistically related fusion architecture. As in class I proteins, a proteolytic cleavage (of PrM to M in flaviviruses, or of pE2 to E2 in alphaviruses) yields mature virions, with the fusion proteins in a metastable conformation, primed for fusion. The fusion peptide, an internal loop at the tip of an elongated subdomain of the protein, is buried at a protein interface and becomes exposed in the conformational change initiated by exposure to low pH.
The mechanism of fusion of class II viral fusion proteins is not well-understood, and there are no therapeutics that can specifically inhibit the fusion of such proteins. Only the pre-fusion structures of one flaviviral and one alphaviral envelope protein have been determined to date. Because fusion is a key step in viral infectivity, a better understanding of the mechanism of class II envelope proteins and identification of druggable regions within such proteins will further development of therapeutics that can specifically inhibit viral infection by flaviviruses, alphaviruses, and hepatitis viruses.